Current Topics : Reviews

Nucleobases are water-soluble compounds that need specific transporters to cross biological membranes. Cumulative evidence based on studies using animal tissues and cells indicates that the carrier-mediated transport systems for purine and pyrimidine nucleobases can be classified into the following two types: concentrative transport systems that mediate nucleobase transport depending on the sodium ion concentration gradient; and other systems that mediate facilitated diffusion depending on the concentration gradient of the substrate. Recently, several molecular transporters that are involved in both transport systems have been identified. The function and activity of these transporters could be of pharmacological significance considering the roles that they play not only in nucleotide synthesis and metabolism but also in the pharmacokinetics and delivery of a variety of nucleobase analogues used in anticancer and antiviral drug therapy. The present review provides an overview of the recent advances in our understanding of the molecular basis of nucleobase transport systems, focusing on the transporters that mediate purine nucleobases, and discusses the involvement of intracellular metabolism in purine nucleobase transport and chemotherapy using ganciclovir.

The retina is a tissue essential for vision, and the blood–retina barrier (BRB) helps to maintain an optimal microenvironment for the neural system in the retina. Recent findings concerning the BRB showed the involvement of transporters at the inner and outer BRB in drug and nutrient transport, suggesting their utility in the development of novel drug delivery systems to the retina. An in vitro–in vivo relationship study of permeability suggested the influx transport of verapamil, a cationic drug, across the BRB, and further in vivo and in vitro studies of cationic drugs, such as verapamil, propranolol and clonidine, revealed the involvement of carrier-mediated process in their influx transport at the BRB. Studies on substrate specificity in TR-iBRB2 cells, an in vitro model cell line of the inner BRB, suggests the involvement of novel organic cation transporter in the influx transport of cationic drugs at the inner BRB. Considering the neuroprotective effect previously reported for several cationic drugs, such as propranolol and clonidine, the study of cation transport at the BRB is widely expected to improve the treatment of retinal diseases, such as diabetic retinopathy and age-related macular degeneration.

Dysfunction in neurotransmission mediated by neurotransmitters causes various neurological disorders. Therefore, receptors and reuptake transporters of neurotransmitters have been focused on as a therapeutic target in neurological disorders. These membrane proteins have high affinity for a specific neurotransmitter and are highly expressed on synaptic membranes. In contrast, xenobiotic transporters have relatively lower affinity for neurotransmitters but widely recognize various organic cations and/or anions and are also expressed in brain neurons. However, it has been largely unknown why such xenobiotic transporters are expressed in neurons that play a key role in signal transduction. We have therefore attempted to clarify the physiological roles of one such xenobiotic organic cation transporter (OCT) in neural cells with the aim of obtaining new insight into the treatment of neurological disorders. Carnitine/organic cation transporter OCTN1/SLC22A4 is functionally expressed in neurons and neural stem cells. In particular, OCTN1 is expressed at much higher levels compared with other OCTs in neural stem cells and positively regulates their differentiation into neurons. OCTN1 accepts the naturally occurring food-derived antioxidant ergothioneine (ERGO) as a good in vivo substrate. Because ERGO is highly distributed into the brain after oral ingestion, OCTN1 may contribute to the alleviation of oxidative stress and promotion of neuronal differentiation via the uptake of ERGO in the brain, perhaps abating symptoms of neurological disorders. In this review, we introduce current topics on the physiological roles of OCTs with a focus on OCTN1 in neural cells and discuss its possible application to the treatment of neurological disorders.

It has become widely acknowledged that astrocytes play essential roles in maintaining physiological central nervous system (CNS) activities. Astrocytes fulfill their roles partly through the manipulation of their plasma membrane transporter functions, and therefore these transporters have been regarded as promising drug targets for various CNS diseases. A representative example is excitatory amino acid transporter 2 (EAAT2), which works as a critical regulator of excitatory signal transduction through its glutamate uptake activity at the tripartite synapse. Thus, enhancement of EAAT2 functionality is expected to accelerate glutamate clearance at synapses, which is a promising approach for the prevention of over-excitation of glutamate receptors. In addition to such well-known astrocyte-specific transporters, cumulative evidence suggests that multi-specific organic ion transporters (i.e., organic cation/anion transporters [OCTs/OATs], carnitine/organic cation transporters [OCTNs], and organic anion transporting polypeptides [OATPs]) are also functionally expressed in astrocytes. Even though identification and characterization of their physiological/pathophysiological roles in astrocytes are in the initial stage, the findings obtained so far indicate that OCT3 and plasma membrane monoamine transporter are significantly involved in the clearance of biogenic amine neurotransmitters in the synaptic cleft, and that OCTN2 and OATP1C1 provide a cellular entry gate for carnitine/acetyl-L-carnitine and thyroxine, respectively. Therefore, organic ion transporters, including those mentioned above, are expected to become emerging pharmacological targets for various CNS diseases. With such expectations in mind, this review will briefly summarize the functional expression of organic ion transporters in astrocytes.

The lack of response to leptin’s actions in the brain, “leptin resistance,” is one of the main causes of the pathogenesis of obesity. However, although high-fat diets affect sensitivity to leptin, the underlying mechanisms of leptin resistance are still an enigma. Here we examined the effect of excess saturated fatty acids (SFAs) on leptin signaling in human neuronal cells. Palmitate, the principle source of SFAs in diet, induced leptin resistance in a human neuroblastoma cell line stably transfected with the Ob-Rb leptin receptor (SH-SY5Y-ObRb). We next investigated the function of stearoyl-CoA desaturase-1 (SCD1), an enzyme which converts SFAs into monounsaturated fatty acids (MUFAs), on leptin-induced signaling. We found that reduction of SCD1 activity, through SCD1 inhibition and knockdown, impairs leptin-induced signal transducer and activator of transcription 3 (STAT3) phosphorylation in human neuronal cells. Our findings suggested that SCD1 plays a key role in the pathophysiology of leptin resistance in neuronal cells associated with obesity.

To develop new pleuromutilin derivatives as veterinary antibiotic medicines, we designed and synthesized a series of new thioether pleuromutilin derivatives possessing acylthiazolyl moiety based on previously designed derivatives. The antibacterial properties of the prepared pleuromutilin derivatives were assessed in vitro by the broth dilution method against five kinds of bacteria and the mycoplasma Mycoplasma gallisepticum (MG). All of the tested compounds displayed moderate to good antibacterial activity to methicillin-sensitive Staphylococcus aureus (MSSA), methicillin-sensitive Staphylococcus epidermidis (MSSE), methicillin-resistant S. aureus (MRSA), Streptococcus agalactiae (S. aga) and MG. However, the activity to Pyogeniccoccus (Pyogens) was generally poor. Compounds 13i and l showed potent antibacterial activity against MSSE and MRSA which are better than that of valnemulin. The structural modification for pleuromutilin affected the antibacterial activity. Amino substituents in the benzene ring can effectively improve activity. Compared with the analogue 13a that possesses unsubstitution benzoyl group, the nitro, methoxy, hydroxy and dichloro substituent contributed little to antibacterial activity. Increasing a methylene between benzene moiety and carbonyl group decreased the bioactivity of derivative. The analogues that obtained by the reaction of amino acids and intermediate 9 showed moderate activity.

Mitochondria are the central hubs for cellular bioenergetics and are crucial to cell survival. It is well accepted that compromised mitochondrial function is linked with hepatocytes injury and contribute to progression of liver diseases. Despite the therapeutic potential of mesenchymal stem cells (MSCs) transplantation on hepatic disorders have been extensively investigated, the effects of MSCs on mitochondrial function in liver injury models remain unknown. Here we investigated the effects of treatment with umbilical cord (UC) MSC in a rat model of D-galactose (D-Gal) induced liver injury, characterized by organ damage, oxidative stress and mitochondrial dysfunction. Our results showed that UC-MSCs treatment significantly alleviated histological lesion and attenuated the elevation of liver biochemical markers, demonstrating its protective effects on D-Gal induced hepatic disorders. Mitochondria isolated from the liver of D-Gal models exhibited decreased antioxidant capacity as well as compromised bioenergetics functions, as shown by a loss of mitochondrial membrane potential, elevation of reactive oxygen species (ROS) production, reduction of mitochondrial respiration complexes and ATP decrement. Treatment of rats with UC-MSCs remarkably blunted these changes and rescued mitochondrial efficiency. Mechanistically, we found that the protective potential of UC-MSCs administration was mediated by nuclear factor-E2-related factor 2/heme oxygenase-1 (Nrf2/HO-1) pathway, but not FOXO3a pathway. In conclusion, the attenuating effects of UC-MSCs on hepatic damage partially rely on normalizing mitochondrial function and preventing a state of energetic deficit via activation of Nrf2/HO-1 pathway.

Exosomes are derived from various sources, including primary and cultured cell lines and body fluids. It is now evident that they are important for communication between cells. They have, therefore, been proposed as potential carriers to deliver drugs to specific sites. In this study, we examined stability of exosomes derived from human saliva. Exosomes were stored at 4°C for up to 20 months and their membrane integrity assessed. Several exosomal markers, such as dipeptidyl peptidase IV (DPP IV; membrane marker) and programmed cell death 6-interacting protein (Alix, lumen marker), were retained intact after 20 months storage at 4°C. Moreover, intact exosomes could be isolated from whole saliva that had been stored at 4°C. Membrane disruption with detergents such as Triton X-100 and Nonidet P-40 caused partial solubilization of DPP IV and release of Alix into the supernatant. In contrast, sodium dodecyl sulfate treatment caused a complete disruption of the membrane. In addition, membrane stability was maintained after freezing and thawing. These results indicated that human saliva-derived exosomes are stable, maintaining their membrane integrity over a long storage period.

Thiazolidinediones (TZDs) are known as peroxisome proliferator-activated receptor γ (PPARγ) activators, and are used in the treatment of diabetes. Although the usefulness of TZDs has been demonstrated, some of their side effects are becoming an obstacle to their clinical applicability; edema is known to be evoked by the “structural characteristics” of TZD, but not by the PPARγ activation. Thus, novel therapeutic modalities (i.e., non-TZD-type PPARγ activators) having different structures to those of TZDs are desired. We previously identified bongkrekic acid (BKA) as a PPARγ activator using the human breast cancer MCF-7 cell line as a model system. In the present study, we newly synthesized BKA analogs and examined the usefulness of BKA and its analogs as PPARγ activators in differentiated adipocyte cells. Among the chemicals investigated, one of the BKA analogs (BKA-#2) strongly stimulated PPARγ and the differentiation of 3T3-L1 cells similar to pioglitazone, a positive control. Furthermore, BKA-#2 reduced the size of lipid droplets in the mature adipocyte cells. The possible modulation mechanism by BKA-#2 is discussed.

The aim of the work was the evaluation of selected biological and physicochemical, applicative properties of ethylated ascorbic acid (AAE) compared to ascorbic acid (AA). Thermogravimetry (TG), differential thermogravimetry (DTG), and differential thermal analysis (DTA) were conducted, followed by the evaluation of AAE decomposition by the UV-Vis spectroscopic method including the influence of temperature and pH. Scavenging, antimicrobial activity and cytotoxicity against L929 fibroblasts were also performed. The difference in mass loss between AA and AAE was 30% via TG. DTA revealed characteristic exothermic and endothermic effects. The AAE solution was more thermally stable than AA. The calculated zero-order rate constants of free-radical scavenging kinetics for AAE were in the range of 4.9×10−3–1.35×10−2 s−1. The activation energy for the process was 11,2281 kJ/mol. AAE was active against Staphylococcus (S.) aureus and Enterococcus(E.) faecalis and acted stronger against Candida(C.) albicans than AA. The concentrations of AA ≥2.5% were cytotoxic, whereas in the case of AAE, a 10% concentration was considered cytotoxic. DTG enables the detailed differentiation between AA and AAE. AAE in aqueous solution is more stable compared to AA. The antioxidant activity of AAE is significant. However, the reaction with 2,2-diphenyl-1-picrylhydrazyl (DPPH) indicates prolonged activity compared to AA. Variability in the antimicrobial activity of AAE may find practical application in the pharmaceutical and cosmetic industries. The potential for applicative aims may be supported by the relatively low in vitro toxicity of AAE.

Luseogliflozin is a selective sodium glucose co-transporter 2 (SGLT2) inhibitor that reduces hyperglycemia in type 2 diabetes mellitus (T2DM) by promoting urinary glucose excretion (UGE). A clinical pharmacology study conducted in Japanese patients with T2DM confirmed dose-dependency of UGE with once-daily administration of luseogliflozin; however, the reason for sustained UGE after plasma luseogliflozin decreased was unclear. To elucidate the effect of inhibition rate constants, Kon and Koff, and to explain the sustained UGE, a pharmacokinetic–pharmacodynamic (PK-PD) model was built based on the mechanisms of glucose filtration in the glomerulus and reabsorption in the renal proximal tubule of kidney as well as the kinetics of competitive inhibition of SGLT1/2 and inhibition rate constants of SGLT2, by using UGE and plasma glucose levels and luseogliflozin concentrations. This acquired population PK-PD model adequately described the sustained UGE and the estimated population means of the inhibition constant for SGLT2 (Ki2) and inhibition-rate constants for SGLT2 (Kon and Koff) were 0.31- and 3.6-fold lower or higher than the in vitro values. Because the dissociation half-time of luseogliflozin from SGLT2 calculated from Koff, 6.81 h, was consistent with the value in vitro, we considered that the sustained UGE could be explained by the long dissociation half-time. Moreover, by calculating the SGLT2 inhibition ratio using the model, we discuss other properties of the UGE time course after luseogliflozin administration.

The aim of study was to establish a mouse model of blue light emitting diode (LED) light-induced retinal damage and to evaluate the effects of the antioxidant N-acetylcysteine (NAC). Mice were exposed to 400 or 800 lx blue LED light for 2 h, and were evaluated for retinal damage 5 d later by electroretinogram amplitude and outer nuclear layer (ONL) thickness. Additionally, we investigated the effect of blue LED light exposure on shorts-wave-sensitive opsin (S-opsin), and rhodopsin expression by immunohistochemistry. Blue LED light induced light intensity dependent retinal damage and led to collapse of S-opsin and altered rhodopsin localization from inner and outer segments to ONL. Conversely, NAC administered at 100 or 250 mg/kg intraperitoneally twice a day, before dark adaptation and before light exposure. NAC protected the blue LED light-induced retinal damage in a dose-dependent manner. Further, blue LED light-induced decreasing of S-opsin levels and altered rhodopsin localization, which were suppressed by NAC. We established a mouse model of blue LED light-induced retinal damage and these findings indicated that oxidative stress was partially involved in blue LED light-induced retinal damage.

Doripenem (DRPM) is a broad-spectrum antibacterial agent often used as empirical therapy for critically ill patients, although there is a lack of studies validating the recommended dosage regimen for patients admitted to intensive care unit (ICU), based on pharmacokinetic (PK)/pharmacodynamic (PD) index. In this study, we estimated the free time above minimum inhibitory concentration (fT>MIC (%)) of DRPM using population PK analysis of 12 patients in ICU, and evaluated the validity of the dosage regimen stratified by creatinine clearance. Using a 2-compartment population PK model reported previously, the mean total clearance or distribution volume of DRPM estimated by Bayesian estimation was significantly lower or higher than that of based on population PK model. The estimated fT>MIC (%) of the recommended standard (normal renal function: 0.5 g every 8 h, moderate: 0.25 g every 8 h, severe renal impairment: 0.25 g every 12 h) and higher doses (normal: 1.0 g every 8 h, moderate: 0.5 g every 8 h, severe: 0.25 g every 8 h) against MICs of 0.5, 1 and 2 µg/mL exceeded 40% in all patients. When stratified by creatinine clearance, the PK/PD breakpoints estimated by Monte Carlo simulation in three grades of renal function tended to be higher than the previously reported PK/PD breakpoints for patients with urinary tract infection, an infection of lesser severity than ICU patients. These results suggest that the dosage regimen stratified by renal function derived from Japanese package insert may be sufficient to achieve effective treatment in ICU patients.

Breast cancer is one of the most lethal malignancies in the world. Oxymatrine is the major effective and toxic alkaloid component which is derived from the root of Sophora flavescens AIT, a traditional Chinese medicine which is widely distributed in Asia and the Pacific Islands. In the current research study, we investigated the effects and mechanisms of action of oxymatrine on breast cancer cells. We demonstrated that the viability and single cell proliferation capability of MCF-7 and MDA-MB-231, two breast cancer cell lines which are widely used in in vitro study, were significantly suppressed in a time- and concentration-dependent manner. Furthermore, the cell cycle of breast cancer cells treated with oxymatrine was arrested at the S-phase of the cell cycle. Oxymatrine also triggered apoptosis in breast cancer cells by modulating apoptosis-related proteins, such as cleaved Caspase-3, cleaved Caspase-9 and poly(ADP-ribose)polymerase (PARP). The remarkable reduction in the ratio of Bcl-2/Bax was also observed in oxymatrine treated breast cancer cells. In conclusion, our research demonstrated that oxymatrine plays a critical role in suppressing carcinogenesis of breast cancer cells through cell cycle arrest and induction of mitochondria-mediated apoptosis, which suggests a promising application of this drug in breast cancer therapy.

Cathepsin B is a lysosomal cysteine protease involved in many diseases. The present research demonstrates that derivatives of epoxysuccinyl–peptide are effective and selective cathepsin B inhibitors. We synthesized a series of epoxysuccinyl–peptide derivatives based on the well-known cathepsin B inhibitor E64d. Specifically, we substituted the 2-methylpropane group at the R1 position of E64d with a sulfane, such as ethyl(methyl) sulfane or benzyl(methyl) sulfane. We also designed and synthesized a library of molecules with various substituents at the R2 position of E64d to replace 2-methylbutane. By studying the structure–activity relationships of these newly synthesized molecules as cathepsin B inhibitors, we demonstrated that substituting ethyl(methyl) sulfane for 2-methylbutane (R2) of E64d improves the inhibitory activity and selectivity for cathepsin B inhibition. Our new cathepsin B inhibitors were highly effective and selective.

Metformin is a commonly used drug for the treatment of type II diabetes and atorvastatin is the most prescribed cholesterol-lowering statin. The present study investigated the effects and mechanisms of metformin and atorvastatin in combination on human prostate cancer cells cultured in vitro and grown as xenograft tumor in vivo. Metformin in combination with atorvastatin had stronger effects on growth inhibition and apoptosis in PC-3 cells than either drug alone. The combination also potently inhibited cell migration and the formation of tumorspheres. Metformin and atorvastatin in combination had a potent inhibitory effect on nuclear factor-kappaB (NF-κB) activity and caused strong decreases in the expression of its downstream anti-apoptotic gene Survivin. Moreover, strong decreases in the levels of phospho-Akt and phosphor-extracellular signal-regulated kinase (Erk)1/2 were found in the cells treated with the combination. The in vivo study showed that treatment of severe combined immunodeficient (SCID) mice with metformin or atorvastatin alone resulted in moderate inhibition of tumor growth while the combination strongly inhibited the growth of the tumors. Results of the present study indicate the combination of metformin and atorvastatin may be an effective strategy for inhibiting the growth of prostate cancer and should be evaluated clinically.

The free fatty acid receptor 1 (GPR40/FFAR1) is activated by polyunsaturated fatty acids (PUFAs) such as docosahexaenoic acids (DHA). This receptor has been the focus of many studies regarding physiological functions of the central nervous system. PUFAs are essential for neuronal development and maintenance of neuronal function; thus, the decrease of PUFAs in the brain is closely related to the induction of psychiatric diseases associated with emotional disorder, such as anxiety, depression, and schizophrenia. However, details of the mechanisms remain unclear. In this study, we investigated changes of maternal and/or emotional behavior caused by a deficiency of GPR40/FFAR1 signaling. GPR40/FFAR1 deficient (FFAR1−/−) female mice exhibited impaired maternal care such as retrieving behaviors and an increased rate of neglect and infanticide when compared to wild type (WT) female mice. Furthermore, FFAR1−/− female mice showed increased time spent in the open arms in an elevated plus maze test, reduction of locomotor activity and social interaction behavior, and decreased sucrose intake, when compared to WT female mice. In conclusion, these findings suggest that PUFAs-GPR40/FFAR1 signaling might function, at least in part, as a regulatory factor of emotional and maternal behavior in mice.

Although magnetic resonance imaging (MRI) has potential in assessments of formulations, few studies have been conducted because of the size and expense of the instrument. In the present study, the processes of in vitro and in vivo release in a gelatin capsule formulation model were visualized using a compact MRI system with 1.5 T permanent magnets, which is more convenient than the superconducting MRI systems typically used for clinical and experimental purposes. A Gd-chelate of diethylenetriamine-N,N,N′,N″,N″-pentaacetic acid, a contrast agent that markedly enhances proton signals via close contact with water, was incorporated into capsule formulations as a marker compound. In vitro experiments could clearly demonstrate the preparation-dependent differences in the release/disintegration of the formulations. In some preparations, the penetration of water into the formulation and generation of bubbles in the capsule were also observed prior to the disintegration of the formulation. When capsule formulations were orally administered to rats, the release of the marker into the stomach and its transit to the duodenum were visualized. These results strongly indicate that the compact MRI system is a powerful tool for pharmaceutical studies.

Dramatic changes in the glycan structures of cell surface proteins have been observed upon malignant transformation of cells as induced by the altered expression levels of glycosyltransferases. Such changes are closely associated with the malignant properties of cancer cells. Transcription factor Sp1 regulates the gene expression of various molecules including glycosyltransferases. Herein, we investigated whether or not Sp1-downregulation affects to N-glycosylation of glycoproteins and malignant properties of A549 human lung cancer cell line. We established a stable clone whose Sp1-expression level was reduced to 50% of a control clone by RNA interference. Lectin blotting revealed that the β4-galactosylation of highly branched N-glycans decreases mainly in cell adhesion molecule, E-cadherin. The analysis of underlying mechanism for decreased β4-galactosylation of N-glycans showed that the gene expression level of β4-galactosyltransferase (β4GalT) 1 decreases dramatically by downregulation of Sp1 without changes in those of β4GalT2 and N-acetylglucosaminyltransferase V. Mutations in the Sp1-binding sites of the β4GalT1 gene promoter showed that the promoter activity decreases significantly, indicating that the gene expression is regulated by Sp1. These results indicate that the β4-galactosylation of highly branched N-glycans decreases by downregulation of Sp1 through the reduced expression of the β4GalT1 gene. Furthermore, the Sp1-downregulated cells showed the suppression of the anchorage-independent growth in soft agar and migratory activity when compared to the control cells. The present study demonstrates that downregulation of Sp1 suppresses the malignant properties of A549 cells through the decreased β4-galactosylation of highly branched N-glycans.

Middle East Respiratory Syndrome Coronavirus (MERS CoV) is a new emerging viral disease characterized by high fatality rate. Understanding MERS CoV genetic aspects and codon usage pattern is important to understand MERS CoV survival, adaptation, evolution, resistance to innate immunity, and help in finding the unique aspects of the virus for future drug discovery experiments. In this work, we provide comprehensive analysis of 238 MERS CoV full genomes comprised of human (hMERS) and camel (cMERS) isolates of the virus. MERS CoV genome shaping seems to be under compositional and mutational bias, as revealed by preference of A/T over G/C nucleotides, preferred codons, nucleotides at the third position of codons (NT3s), relative synonymous codon usage, hydropathicity (Gravy), and aromaticity (Aromo) indices. Effective number of codons (ENc) analysis reveals a general slight codon usage bias. Codon adaptation index reveals incomplete adaptation to host environment. MERS CoV showed high ability to resist the innate immune response by showing lower CpG frequencies. Neutrality evolution analysis revealed a more significant role of mutation pressure in cMERS over hMERS. Correspondence analysis revealed that MERS CoV genomes have three genetic clusters, which were distinct in their codon usage, host, and geographic distribution. Additionally, virtual screening and binding experiments were able to identify three new virus-encoded helicase binding compounds. These compounds can be used for further optimization of inhibitors.

Ions, small molecules, and drugs are absorbed in the intestinal epithelium mediated by transcellular and paracellular pathways. The function of various transporters expressing in the apical and basolateral membranes of intestinal epithelial cells has been well characterized. In contrast, claudins and occludin, components of the tight junctions (TJs), determine the paracellular permeability to ions and low molecular weight compounds, but the properties for permeability has not been clarified in detail. In the present study, we examined the effects of anti-histamine drugs, chlorpheniramine and diphenhydramine, on transepithelial electrical resistance (TER) and permeability to lucifer yellow (LY), a marker of paracellular permeability, using murine colonic MCE301 cells. Chlorpheniramine significantly decreased the steady state of TER and increased permeability to LY, whereas the effects of diphenhydramine were not significant. The mRNAs of occludin and claudin-1-claudin-8 except for claudin-5 were expressed in MCE301 cells. Both anti-histamine drugs did not change solubility of claudins to 0.5% Triton X-100 solution. In contrast, the detergent solubility and intracellular localization of occludin were significantly increased by chlorpheniramine. These results indicate that occludin is dissociated from the TJs by chlorpheniramine. Chlorpheniramine increased protein phosphatase-2A (PP-2A) activity, which was inhibited by cantharidin, a potent PP-2A inhibitor. Furthermore, the changes of TER, permeability to LY, and de-phosphorylation and tight junctional localization of occludin caused by chlorpheniramine were recovered by cantharidin. These results suggest that chlorpheniramine could increase paracellular permeability to low molecular weight compounds mediated by the activation of PP-2A and internalization of occludin in the colonic epithelial cells.

Tadalafil and sildenafil are selective inhibitors of phosphodiesterase type 5, showing marked pharmacokinetic variability in patients with pulmonary arterial hypertension. It has been reported that sildenafil is a substrate for P-glycoprotein (P-gp), but whether tadalafil is a substrate for P-gp remains to be determined. The objective of the present study was to elucidate whether tadalafil is a substrate for P-gp. Transcellular transport of sildenafil and tadalafil (5 µM each) was examined using renal epithelial LLC-PK1 and P-gp-expressing LLC-GA5-COL150 cell monolayers. The efflux ratio of the basal to apical (B to A) transport of sildenafil to the A to B transport after 120-min incubation in LLC-GA5-COL150 cells (1.52) was significantly higher than that in LLC-PK1 cells (0.711). The efflux ratio of the B to A transport of tadalafil to the A to B transport after 120-min incubation in LLC-GA5-COL150 cells (10.4) was significantly higher than that in LLC-PK1 cells (1.23). In LLC-GA5-COL150 cell monolayers, the Vmax and Km values of sildenafil transport calculated from a modified Michaelis–Menten equation were 101±64 pmol/min/cm2 and 112±47 µM, respectively. On the other hand, those of tadalafil transport were 13.6±4.8 pmol/min/cm2 and 22.7±9.3 µM, respectively. In the presence of a P-gp inhibitor (PSC833), the B to A transport of tadalafil was decreased by 28.6% in LLC-GA5-COL150 cells, and the A to B transport of tadalafil was 6.59-fold greater than that in its absence. These results indicate that tadalafil is a substrate for P-gp.

The effects of zebularine, a DNA methyltransferase inhibitor, on the invasion activity as well as intracellular expression level of let-7b, tumor suppressor microRNA, were examined in three human colorectal cancer (CRC) cell lines: SW480, SW620, and oxaliplatin-resistant SW620 (SW620/OxR). Zebularine suppressed the invasion activity of SW620 and SW620/OxR cells. The intracellular expression level of let-7b was up-regulated by zebularine in SW620 and SW620/OxR cells. The overexpression of let-7b by the transfection of let-7b mimic suppressed invasion activity in SW620 and SW620/OxR cells. These results suggest that zebularine may inhibit invasion activity by up-regulating the intracellular expression level of let-7b in high-invasive CRC cells.

Recent studies suggest that histamine—a regulator of the microcirculation—may play important roles in exercise. We have shown that the histamine-forming enzyme histidine decarboxylase (HDC) is induced in skeletal muscles by prolonged muscular work (PMW). However, histological analysis of such HDC induction is lacking due to appropriate anti-HDC antibodies being unavailable. We also showed that the inflammatory cytokines interleukin (IL)-1 and tumor necrosis factor (TNF)-α can induce HDC, and that PMW increases both IL-1α and IL-1β in skeletal muscles. Here, we examined the effects (a) of PMW on the histological evidence of HDC induction and (b) of IL-1β and TNF-α on HDC activity in skeletal muscles. By immunostaining using a recently introduced commercial polyclonal anti-HDC antibody, we found that cells in the endomysium and around blood vessels, and also some muscle fibers themselves, became HDC-positive after PMW. After PMW, TNF-α, but not IL-1α or IL-1β, was detected in the blood serum. The minimum intravenous dose of IL-1β that would induce HDC activity was about 1/10 that of TNF-α, while in combination they synergistically augmented HDC activity. These results suggest that PMW induces HDC in skeletal muscles, including cells in the endomysium and around blood vessels, and also some muscle fibers themselves, and that IL-1β and TNF-α may cooperatively mediate this induction.

Eleven kinds of catechin metabolites produced from (−)-epigallocatechin (EGC) and (−)-epigallocatechin gallate (EGCg) by intestinal microbiota were evaluated for inhibitory activity on the proliferation of HeLa cells, which are human cervical cancer cells. Among the catechin metabolites, 1-(3,4,5-trihydroxyphenyl)-3-(2,4,6-trihydroxyphenyl)propan-2-ol (EGC-M2), 4-hydroxy-5-(3,4,5-trihydroxyphenyl)valeric acid (EGC-M7), and 5-(3,4,5-trihydroxyphenyl)valeric acid (EGC-M9) were found to show inhibitory activity on HeLa cell proliferation as compared with control. The results suggested that three adjacent hydroxyl groups in the phenyl moiety may play an important role in the inhibitory activity. In addition, the inhibitory activity was also examined with four (−)-epicatechin (EC) metabolites possessing two adjacent hydroxyl groups in the phenyl moiety. Only 5-(3,4-dihydroxyphenyl)valeric acid (EC-M9) showed inhibitory activity and therefore valeric acid moiety likely contributes to the inhibitory activity. EGC-M9 showed the strongest inhibitory activity with IC50 of 5.58 µM. Thus, in this study it was found for the first time that several catechin metabolites derived from EGC, EGCg, and EC inhibit the proliferation of cervical cancer cells.

System Maintenance

Announcement From J-STAGE

July 31, 2017Due to the end of the Yahoo!JAPAN OpenID service, My J-STAGE will end the support of the following sign-in services with OpenID on August 26, 2017: -Sign-in with Yahoo!JAPAN ID -Sign-in with livedoor ID * After that, please sign-in with My J-STAGE ID.

July 03, 2017There had been a service stop from Jul 2‚ 2017‚ 8:06 to Jul 2‚ 2017‚ 19:12(JST) (Jul 1‚ 2017‚ 23:06 to Jul 2‚ 2017‚ 10:12(UTC)) .The service has been back to normal.We apologize for any inconvenience this may cause you.

May 18, 2016We have released “J-STAGE BETA site”.

May 01, 2015Please note the "spoofing mail" that pretends to be J-STAGE.